Synthesis, cytotoxicity and
antiplasmodial activity of novel
ferrocenyl-artemisinin hybrids
C de Lange
orcid.org / 0000-0002-4252-5433
Thesis submitted in fulfilment of the requirements for the degree
Doctor of Philosophy in Pharmaceutical Sciences
at the
North-West University
Promotor:
Prof DD N‘Da
Co-Promotor:
Prof RK Haynes
Additional Co-Promotor:
Dr FJ Smit
Graduation: October 2018
Student number: 20256353
ii
Preface
This thesis is submitted in an article format in accordance with the General Academic Rules (A) of the North-West University. Three articles, two of which have been published, are included in this thesis:
Chapter 3: Article 1
Non-acetal artemisinin derivative – Worth the fuss? A mini-review. Intended to be submitted
to Current Medicinal Chemistry Journal. Chapter 4: Article 2
Synthesis, in vitro antimalarial activities and cytotoxicities of amino-artemisinin-ferrocene derivatives. This article was published in Bioorganic & Medicinal Chemistry Letters, Volume 28, Issue 3, 1 February 2018, Pages 289-292 (https://doi.org/10.1016/j.bmcl.2017.12.057).
Chapter 5: Article 3
Synthesis, in vitro antimalarial activities and cytotoxicity of amino-artemisinin-1, 2-disubstituted ferrocene derivatives. The article was published in Bioorganic and Medicinal Chemistry Letters, Volume 28, Issue 19, 15 October 2018, Pages 3161-3163 (https://doi.org/10.1016/j.bmcl.2018.08.037).
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Acknowledgements
I hereby wish to express my appreciation to the following individuals: Prof DD N‘Da, my Promoter, for his insight, encouragement and support throughout the study.
Prof RK Haynes, my Co-Promoter, for his insights and ideas throughout the study. Dr FJ Smit, my additional Assistant Promoter, for his assistance in the laboratory and
the countless hours spend revising my work.
Dr JHL Jordaan, for his consistent support and for the collection of MS data. Mr A Joubert, for the collection of the NMR data.
Dr HN Wong, for her assistance in the laboratory.
Prof L Birkholtz and Dr D Coertzen from UP, for the antimalarial screening of the derivatives.
Dr JF Wentzel, for the cytotoxicity and anticancer screening of the derivatives. The NRF, MRC and the North-West University, for financial support.
Prof S van Dyk, Director of the School of Pharmacy, for her support.
Prof A Wessels and Prof G Terre‘ Blanche for allowing me to be a demonstrator for them.
All of my friends that encouraged and kept me going when times got tough. My family for their support and believe in me throughout the whole process. My loving wife for her constant encouragement and love.
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Abstract
Malaria is caused by a parasite of the genus Plasmodium. Of the five species that infect humans, Plasmodium falciparum is the most dangerous. This disease caused 435 thousand deaths in 2017. It is estimated that 266 thousand of these deaths were children, under the age of five. With the reports of malaria resistance towards artemisnin, there is a desperate need for new and effective antimalarial drugs. In the search for these new antimalarial drugs, two series of artemisinin-ferrocene derivatives were prepared and investigated during this study.
A mini-review (Chapter 3) was written in order to compare the most potent non-acetal artemisinin derivatives. In order to compare these derivatives, relative activity was used. Due to the large variety of Plasmodium falciparum strains used it is difficult to truly compare these derivatives. The general lack of toxicity data for these derivatives makes it difficult to establish whether the activity is due to toxicity. The logP value was calculated for these derivatives to be able to estimate toxicity. It was shown that there is some connection between lipophilicity and toxicity.
The first series (Chapter 4) of amino-ferrocene-artemisinin derivatives was synthesized by the coupling of various mono-substituted ferrocene derivatives to 10α-(1′-piperazino)-10-deoxo-10-dihydroartemisinin (DHA-pip) through condensation and reductive amination. These derivatives were screened against the chloroquine-sensitive (CQ-sensitive) NF54 and chloroquine-resistant (CQ-resistant) K1 and W2 P. falciparum strains. Cytotoxicity was assessed against the Hek293 cell line while anticancer activity was assessed against the A375 cell line. The derivatives retained good antimalarial activity while being very selective towards parasitized cells in the presence of mammalian cells. Additionally these derivatives were in general more selective towards cancer cells in the presence of mammalian cells.
The second series (Chapter 5) of amino-artemisinin-1, 2-disubstituted ferrocene derivatives was synthesized through reductive amination of aminoferrocenealdehydes to DHA-pip. These derivatives were screened against the CQ-sensitive NF54 and CQ-resistant K1 and W2 P. falciparum strains. Cytotoxicity was assessed against the Hek293 cell line while anticancer activity was assessed against the A375 cell line. These derivatives were also screened against P falciparum NF54 gametocytes. Two of these derivatives were more active than DHA while the activity of one of these derivatives might be attributed to toxicity.
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The least antimalarial active derivative was more active and selective towards cancer cells in the presence of mammalian cells.
This study resulted in a number of derivatives with different antiplasmodial activities. The derivatives of series 2 were the most active due to the single ring disubstituted ferrocene derivatives. The derivatives that were synthesized during the study illustrate a low potential for resistance and addresses the problem of P. falciparum. These derivatives could potentially serve as lead compounds for future antimalarial drugs.
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Opsomming
Malaria word deur ‘n parasiet uit die genus Plasmodium veroorsaak. Uit die vyf spesies wat mense kan besmet is Plasmodium falciparum die gevaarlikste. Hierdie siekte het omtrent 435 duisend sterftes veroorsaak in 2017. Dit word beraam dat 266 duisend van hierdie sterftes was kinders onder die ouderdom van vyf jaar. Met verslae van weerstandbiedigheid teen artemisiniene, is daar ‘n noodsaaklikheid vir nuwe en effektiewe anti-malariamiddels. In die soektog na hierdie anti-malariamiddels, is twee artemisinien-ferroseen reekse verbindings tydens hierdie studie gesintetiseer en ondersoek.
‘n Mini-oorsig (Hoofstuk 3) was geskryf om die mees kragtigste nie-asetaal artemisinien-verbindings te vergelyk. Ten einde hierdie afgeleides te vergelyk, is relatiewe aktiwiteit gebruik. As gevolg van die groot verskeidenheid Plasmodium falciparum stamme, is dit moeilik om hierdie verbindings werklik te vergelyk. Die algemene tekort aan toksisiteitsdata vir hierdie verbindings maak dit moeilik om vas te stel of hierdie aktiwiteit as gevolg van toksisiteit is. Die logP waarde is bereken vir hierdie verbindings om die toksisiteit te skat. Daar was getoon dat daar ‘n verband bestaan tussen lipofilisiteit en toksisiteit is. Daar is bevind dat die gebrek aan vergelykbaarheid en toksisteit die ideale teen-malariamiddel weerhou om ooit gesintetiseer te word.
Die eerste reeks verbindings (Hoofstuk 4) het aminoferroseniel-artemisiniene behels, wat deur middle van kondensasie en reduktiewe aminering van verskeie ferrosiniel intermediêre met 10α-(1′-piperasienniel)-10-deoksie -10-dihdroartemisinien (DHA-pip) gesintetiseer is. Hierdie verbindings is teen die CQ-sensitiewe NF54 en die CQ-weerstandige K1 en W2 P.
falciparum stamme getoets. Sitotoksisiteit is geassesseer teen die Hek293 sellyn terwyl die
kankeraktiwiteit teen die A375 sellyn geassesseer is. Die verbindings het goeie teen-malaria aktiwiteit behou terwyl hulle baie selektief was teenoor parasiete in die teenwoordigheid van soodier selle. Daarbenewens was hierdie verbindings meer selektief teenoor kankerselle in die teenwoordigheid van soogdier selle.
Die tweede reeks (Hoofstuk 5) het amino-artemisinien 1,2-digesubstitueerde ferroseniel verbindings behels, wat deur reduktiewe aminering van aminoferrosenielalhiede en DHA-pip gesintetiseer was. Hierdie verbindings is teen die sensitiewe NF54 en die CQ-weerstandige K1 en W2 P. falciparum stamme getoets. Sitotoksisiteit is geassesseer teen die Hek293 sellyn terwyl die teen-kankeraktiwiteit teen die A375 sellyn geassesseer is.
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Hierdie verbindings is ook teen P. falciparum NF54 gametosiete getoets. Twee van hierdie verbindings was meer aktief as dihidroartemisiniel alhoewel een van hierdie verbindings se aktiwiteit aan toksisiteit toegeskryf kan word. Die verbinding wat die minste aktief was teenoor malaria was weer meer aktief en selektief teenoor kankerselle in die teenwoordigheid van soogdierselle.
Hierdie studie het gelei tot ‘n aantal verbindings met verskillende antiplasmodiale aktiwiteite. Die verbindings van reeks 2 was die mees aktiefste weens die enkelring digesubstitueerde ferroseniel verbindings. Die verbindings wat tydens hierdie studie gesintetiseer was toon ‘n lae potensiaal vir weerstand vorming en spreek die probleem van weerstandige P.
falciparum aan. Hierdie verbindings is potensiële leidingverbindings om as toekomstige
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List of Contents
Solemn Declaration ... xvii
Preface... ...ii
Letter of agreement... ... iii
Acknowledgements...iv
Abstract...v
Opsomming... ... xvii
List of Contents... xii
List of Figures... ... ...xii
List of Tables and Schemes...xiv
List of Abbreviations ... xvi
Chapter 1: Introduction and Problem Statement... 1
1.1 Background ... 1
1.2 Aim and objectives ... 6
1.3 References ... 8
Chapter 2: Literature review ... 15
2.1 Introduction... ... 15
2.2 Epidemiology ... 16
2.3 The life-cycle of malaria ... 18
2.3.1 Human liver stage ... 19
2.3.2 Human blood stage ... 19
2.3.3 Sporogonic cycle ... 20
2.4 Pathology... ... 20
2.5 Diagnosis... ... 21
2.6 Control and prevention ... 21
x
2.7.1 Quinoline and quinoline based antimalarials ... 23
2.7.1.1 Aryl-amino alcohols ... 23 2.7.1.2 4-Aminoquinolines ... 25 2.7.1.3 8-Aminoquinolines ... 27 2.7.2 Hydroxynaphthoquinones ... 28 2.7.3 Antifolates... 28 2.7.4 Antibiotics ... 31 2.7.5 Artemisinin ... 33 2.7.5.1 Introduction ... 33 2.7.5.2 Mechanisms of action ... 33
2.7.5.3 Artemisinin and its first generation semisynthetic peroxides ... 37
2.7.6 Artemisinin combinational therapy (ACT) ... 38
2.7.7 Resistance towards artemisinin ... 39
2.8 Ferrocene... ... 40
2.8.1 Introduction ... 40
2.8.2 Ferrocene pharmacophore ... 42
2.8.3 Ferrocene artemisinins ... 44
2.8.4 Other artemisinin derivatives ... 46
2.9 Summary... 48
2.10 References ... 49
Chapter 3: Non-acetal artemisinin derivatives – Worth the fuss? A mini-review – Article 1... 78
3.1 Introduction... ... 79
3.2 Synthesis and antimalarial activity ... 82
3.2.1 Group A derivatives ... 82
3.2.2 Group B derivatives ... 85
3.2.3 Group C derivatives ... 86
xi
3.2.5 Group E derivatives ... 92
3.3 In vivo activity and cytotoxicity ... 95
3.4 Conclusion... ... 96
3.5 References... ... ...99
Chapter 4: Synthesis, in vitro antimalarial activities and cytotoxicities of amino- artemisinin-ferrocene derivatives ... 106
Chapter 5: Synthesis, in vitro antimalarial activities and cytotoxicities of amino- artemisinin-1, 2-disubstituted ferrocene derivatives ... 111
Chapter 6: Summary and conclusion ... 115
6.1 References... ... 117
Addendum A: Analytical data for Chapter 4 ... 120
xii
List of figures
Figure 1.1 Artemisinin and the clinically used artemisinins ... 3
Figure 1.2 Structures of chloroquine and ferroquine ... 4
Figure 1.3 Ferrocenyl artemisinin derivatives ... 5
Figure 1.4 Non acetal derivatives ... 6
Figure 2.1 Malaria vector species feeding on humans and animals ... 17
Figure 2.2 Worldwide distribution of P. falciparum ... 17
Figure 2.3 Malaria life cycle ... 18
Figure 2.4 Quinine ... 23 Figure 2.5 Mefloquine ... 24 Figure 2.6 Halofantrine ... 24 Figure 2.7 Lumefantrine ... 25 Figure 2.8 Chloroquine (CQ) ... 25 Figure 2.9 Amodiaquine ... 26 Figure 2.10 Piperaquine ... 27 Figure 2.11 Primaquine ... 27 Figure 2.12 Atovaquone ... 28 Figure 2.13 Dapsone ... 29 Figure 2.14 Sulfalene ... 30 Figure 2.15 Sulfadoxine ... 30
Figure 2.16 Proguanil and metabolite cycloguanil ... 30
Figure 2.17 Chloroproguanil and metabolite chlorocycloguanil ... 31
Figure 2.18 Pyrimethamine ... 31
Figure 2.19 Tetracycline ... 32
Figure 2.20 Doxycycline ... 32
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Figure 2.22 Artemisinin and the clinically used artemisinins ... 37
Figure 2.23 HOMO and LUMO of ferrocene ... 41
Figure 2.24 Ferrocene derivatives ... 43
Figure 2.25 Ferrocenyl artemisinin derivatives of the group of Paitayatat. ... 44
Figure 2.26 Ferrocenyl artemisinin derivatives synthesized by the group of Delheas. ... 45
Figure 2.27 Ferrocenyl artemisinin derivatives of the group of Reiter ... 45
Figure 2.28 Artemisone metabolites ... 47
Figure 3.1 Artemisinin derivatives ... 80
Figure 3.2 Benzyldeoxoartemisinin... 83
Figure 3.3 Chloroquine linkers 23 and 24 ... 91
Figure 3.4 Derivative 25 synthesized by Chadwick et al. (Chadwick et al., 2010) ... 91
xiv
List of tables and schemes
Scheme 1.1 Redox cycling reactions of ferrocene ... 5
Scheme 2.1 Folic acid synthesis pathway ... 29
Scheme 2.2 Antioxidant system ... 36
Scheme 2.3 Preparation of ferrocene ... 41
Scheme 2.4 Synthesis of artemisone ... 46
Scheme 3.1 Grouping of the non-acetal derivatives based on starting material with the yield from artemisinin in brackets ... 82
Scheme 3.2 Synthesis of Deoxyartemisinin (9) from 1 ... 83
Scheme 3.3 Furan deoxoartemisinin derivative, 11 ... 84
Scheme 3.4 Synthesis of 12 ... 84
Scheme 3.5 Synthesis of 13 ... 85
Scheme 3.6 Synthesis of the new 14 ... 86
Scheme 3.7 Pyrrole mannich base derivative 15 and 16 ... 86
Scheme 3.8 Synthesis of 5 ... 87
Scheme 3.9 Synthesis of 17 ... 87
Scheme 3.10 3,3-dimethyl-2-butanol derivative 18 from 17 ... 88
Scheme 3.11 Synthesis of 19 a-c by Khac et al. ... 88
Scheme 3.12 Artemisinin and primaquine hybrid 20 ... 89
Scheme 3.13 Synthesis of 12β-(2-hydroxyethyl)deoxoartemisinin by Araújo et al ... 89
Scheme 3.14 10β-[2-(2-Fluorobenzyloxy)ethyl]deoxoartemisinin (21)with a yield of 16% from 1 ... 90
Scheme 3.15 Synthesis of 22 ... 90
Scheme 3.16 Biotin derivative 26 ... 92
Scheme 3.17 12-n-Butyldeoxoartemisinin (27) synthesis ... 93
Scheme 3.18 Synthesis of 28. ... 93
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Scheme 3.20 Synthesis of derivative 30 ... 94
Table 3.1 Relative activities and ClogP values of derivatives ... 96
xvi
List of abbreviations
°C degrees Celsius
µL microlitre
ACT artemisinin combination therapy
AM artemether
ART artemisinin
AS artesunate
ATP Adenosine triphosphate
CNS Central nervous system
Cp cyclopentadiene CQ chloroquine DDT Dichlorodiphenyltrichloroethane DHA dihydroartemisinin DHF dihydrofolate DHFR dihydrofolate reductase
dhfr-ts Dihydrofolate Reductase-Thymidylate. Synthase
DHFS dihydrofolate synthase
DHP dihydropteroate
DHPP dihydropteridine phosphate
DHPS dihydropteroate synthase
DIAD Diisopropyl azodiformate
DNA Deoxyribonucleic acid
FAD flavin adenine dinucleotide
FADH2 reduced flavin adenine dinucleotide
fc ferrocene FMN flavin mononucleotide FQ ferroquine G6PD glucose-6-phosphate dehydrogenase GR glutathione reductase GSH glutathione GSH-Px glutathione-dependent peroxidise GSSG glutathione disulfide h hour
xvii
HRMS high resolution mass spectroscopy
i-BuLi iso-butyllithium
IC50 half maximal inhibitory concentration
IR infrared
IRS indoor residual spraying
ITN insecticide treated nets
Kd dissociation constant
logP a measure of lipophilicity
LUMO Lowest unoccupied molecular orbital
NADP+ oxidised NADPH
NADPH Nicotinamide adenine dinucleotide phosphate
n-BuLi normal-butyllithium
nM nanomolar
NMR nuclear magnetic resonance
pABA para-amino benzoic acid
Pf Plasmodium faciparum
PfATP6 Plasmodium falciparum Ca(2+)-ATPase
PfCRT Plasmodium falciparum chloroquine resistance transporter
PfMDR1 Plasmodium falciparum multidrug-resistance gene 1
PfNHE1 Plasmodium falciparum Na+/H+ Exchanger (Pfnhe-1) Gene
PPh3 Triphenylphosphine
PVC polyvinyl chloride
RBC red blood cell
RDT rapid diagnostic test
ROS reactive oxygen species
SERCA sarco-endoplasmic reticulum membrane calcium ATPase
SOD superoxide dismutase
t-BuLi tert-Butyllithium THF tetrahydrofuran Trx(S)2 thioredoxin Trx(SH)2 Reduced thioredoxin Trx-Px thioredoxin-dependent peroxidise UV ultra violet
1
Chapter 1:
Introduction and Problem Statement
1.1
Background
According to the World Health Organization (WHO), there were approximately 435 000 deaths in 2017. Of these deaths an astounding 92 % occurred in Africa, of which 61 % were children under the age of five (WHO, 2018). Malaria is a disease caused by an intercellular parasite of the genus Plasmodium. Humans can be infected with malaria by the following species: P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax (Cox et al., 2008). Malaria cannot be transmitted at temperatures below 20°C or above 35°C and as water is needed for mosquitoes to breed it makes sense that malaria is predominant in tropical and subtropical regions (Wernsdorfer, 2012). Additionally some of the poorest and least– developed countries fall in these regions making the availability of resources and logistics needed for malaria prevention and cure cumbersome.
The geographical distribution of Plasmodia species varies. P. falciparum is the dominant species in sub-Sahara Africa and warmer regions of Asia and South America (Gething et al., 2011). P. vivax on the other hand is not as sensitive to cool temperatures and is dominant in India and South America. The low prevalence of P. vivax and P. knowlesi in the African continent is due to the lack of the Duffy antigen in the black African community (Cutbush et
al., 1950; Miller et al., 1976; Neote et al., 1994). This antigen is located on the surface of the
red blood cells and the lack of this antigen leads to a natural resistance towards P. vivax and
P. knowlesi to the individuals. Other erythrocyte disorders that grant the individual malaria
immunity is Sickle cell anaemia and glucose-6-phosphate dehydrogenase deficiency (Ayi et
al., 2004; Williams, 2006). P. knowlesi is a malaria parasite found in long-tailed macaque
monkeys and is transmitted to humans, mainly distributed through South East Asia (Cox-Singh et al., 2008; (Cox-Singh et al., 2004). P. malariae and P. ovale is found in sub-Sahara Africa, Papua New Guinea and in South East Asia (Boutin et al., 2005; Mehlotra et al., 2000; Win et al., 2002).
As a person is infected with malaria there is an incubation period before the onset of symptoms. Initially the symptoms of malaria manifest itself as headache, slight fever, muscle
2
pain and nausea similar to flu symptoms (Schlagenhauf & Steffen, 1994). As the infection progresses, it is followed by fevers due to the rupturing of eurythrocytes (James et al., 1936). It is known that P. vivax causes a number of deaths but P. falciparum is the leading cause of malaria related deaths (Bartoloni & Zammarchi, 2012). The rapid reproduction of P.
falciparum leads to high levels of parasitemia in a short amount of time (Newby et al., 2008).
In the majority of the cases it leads to severe malaria (Jakeman et al., 1999). The most dangerous complications that can develop are cerebral malaria and severe anaemia (Goldsmith, 1997). Although this is the standard route of the manifestation of malaria symptoms there are some variations between species. It was found that P. falciparum, P.
vivax, P. ovale and P. malariae are all capable of asymptomatic infections (Alves et al.,
2002; Rojo-Marcos et al., 2011; Vinetz et al., 1998). Moreover some of the P. vivax and P.
ovale parasites are able to become dormant and cause a relapse of malaria months or years
after the initial infection (Cogswell, 1992). These two species are at greater risk of developing resistance due to relapse of malaria (Farooq & Mahajan, 2004).
It was reported by the WHO that the frequency of malaria infections dropped by 21% between 2010 and 2015. During this time the fatalities also decreased globally by 29%. These statistics were obtained by the increased efforts of the WHO to distribute insecticide treated bed nets and applying indoor residual spraying. Further to this the increased use of rapid diagnostic testing enabled physicians to rapidly distinguish between malaria and non-malaria fevers. The most effective treatment of P. falciparum is artemisinin based combinational therapy. This strategy was formulated by the WHO in order to protect the artemisinin class preventing it from falling victim to P. falciparum resistance. But alas, despite all of these efforts, there is clear evidence of artemisinin resistance.
P. falciparum has grown resistant towards chloroquine, sulfadoxine and pyrimetamine,
mefloquine, atovaquone and proguanil, artemether and lumefantrine (Dondorp et al., 2009; Fivelman et al., 2002; Gregson & Plowe, 2005; Payne, 1987; Price et al., 2004). The most effective treatment left for P. falciparum is the artemisinin class (Figure 1.1) (Graham et al., 2010). Artemisinin (qinghaosu) is a sesquiterpene lactone extracted from sweet wormwood (Artemisia annua) (Klayman, 1985). Uncomplicated cases of malaria should be treated with artemisinin combination therapies (ACTs) while severe malaria should be treated with artesunate (Dondorp et al., 2010).
3
Figure 1.1: Clinically used artemisinins.
The mechanism of action of artemisinins is widely debated but the co-factor theory seems to be unifying (Haynes et al., 2012). As the parasite exists in an oxidative–stressed environment it is crucial to maintain the redox homeostasis. This is achieved by the glutathione:glutathione disulfide (GSH:GSSG) ratio (Ursini et al., 2016). GSH is needed by enzymes to convert damaging substances such as peroxides to water and oxygen, GSH in turn is reduced to GSSG. To convert GSSG back to GSH the flavoenzyme, glutathione reductase (GR) catalyses the reaction using nicotinamide adenine dinucleotide phosphate (NADPH) (Färber et al., 1998). It was found that when yeast GR was treated with artemisinin, an increased consumption of NADPH and decreased the GSSG reduction was observed (Haynes et al., 2010). Additionally GSSG can also be converted to GSH by thioredoxin which is flavin adenine dinucleotide (FADH2) dependent (Jortzik & Becker, 2012). It was found by Haynes and co-workers that artemisinins oxidize reduced FADH2, reduced flavin mononucleotide (FMNH2), reduced riboflavin and model reduced flavins (Haynes et al., 2010). The FADH2 required by the parasites‘ redox system to function optimally are consumed by artemisinin. This leads to enhanced turnover of NADPH - eventually a choke point is reached wherein the requirements by the enzyme for NADPH exceeds the supply.
In 2002 the first sign of ACT resistance was observed with a decline in efficacy for artemisinin-mefloquine treatment (Denis et al., 2002). Fourteen years later the ACT of dihydroartemisinin-piperaquine did not cure half of the patients treated (Fairhurst & Dondorp, 2016). This observation was made in Western-Cambodia, an area known for the formation of resistance towards previously used antimalarials. Moreover, the WHO identified several sites with suspected or confirmed artemisinin resistance. On the Cambodian border, the Vietnamese province of Binh Phuoc reported a higher than 10% ACT failure rate (WHO, 2016). The common metabolite of the clinically used artemisinin, which might be implicated in artemisinin resistance, is dihydroartemisinin (Davis et al., 2005; Mbengue et al., 2015; Paloque et al., 2016). Dihydroartemisinin has a short elimination half–life of 1.9 hrs and is
4
stable in simulated stomach acid for 17 hrs (Jung & Lee, 1998; Teja-isavadharm et al., 1996). The elimination half–life can be addressed through hybridisation with a longer acting pharmacophore while the metabolite dihydroartemisinin can be avoided through non-acetal derivatives.
A novel approach to address resistance is by developing hybrid drugs (Walsh & Bell, 2009). This is achieved by combining two pharmacophores via a chemical bond (Meunier & Vásquez, 2008). These pharmacophores should have different biological functions and by combining them into one hybrid drug the activity should be better than the individual components. Hybrid drugs can interact with a target in one of three different ways. Firstly the two targets are related to one another and the hybrid drug interacts with both of them simultaneously. Secondly targets are in different organelles and the hybrid drug interacts independently. Lastly both the pharmacophores of the hybrid drug has the same target.
One of the leading examples of a hybrid drug that overcame resistance is ferroquine; ferrocene incorporated into the structure of chloroquine (Figure 1.2) (Biot et al., 1997). The position of ferrocene within chloroquine is important as ferroquine was the most active of the more than 50 chloroquine-ferrocene compounds screened (Dive & Biot, 2008). A contributing factor to the activity of ferroquine is that ferrocene has the ability to undergo redox reactions and generate reactive oxygen species (ROS) whereas chloroquine cannot (Dubar et al., 2008).
Figure 1.2: Structures of chloroquine and ferroquine.
Upon metabolism the ferrocenes‘ Fe2+ centre is capable of acting as a redox centre that undergoes redox cycling (Scheme 1.1). Ferrocene could be oxidized by free or labile Fe3+ to form ferrocenium (ferrocene-Fe3+) (Dubar et al., 2011; Kitaguchi & Yoshimura, 2010; Pladziewicz & Espenson, 1973). The newly generated free or labile Fe2+ is oxidized by oxygen to form superoxide which could then form hydroxyl radicals via the Fenton pathway. This increases the ROS which interrupts the redox homeostasis of the parasite. Ferrocenium in turn is reduced to ferrocene by metalloproteins (ferrocytochrome c), NADH and thiols such
5
as GSH (Carlson & Miller, 1983; Matsue et al., 1987; Pladziewicz et al., 1985; Pladziewicz & Carney, 1982). Unfortunately, these enzymes are only present in vivo and the full potential of the compounds will only become apparent in animal studies.
Scheme 1.1: Redox cycling reactions of ferrocene.
Although the mechanisms by which artemisinins exert their antimalarial activity involve ROS, thus this feature will be enhanced by linking ferrocene to the artemisinin structure. Various groups studied the effect of incorporating a ferrocene moiety onto the artemisinin structure with only one derivative showing promise. The group of Delheas synthesized an amine-containing ferrocenyl artemisinin derivative (Figure 1.3) and was the most potent towards chloroquine resistant P. falciparum (Delhaes et al., 2000). An IC50 value of 14 nM was obtained against the Dd2 strain while artemisinin had a value of 13 nM. Unfortunately as many other derivatives it lacks toxicity data.
Figure 1.3: Ferrocenyl artemisinin derivative.
The metabolite of all the clinically used artemisinins is dihydroartemisinin (Figure 1.1) which is linked to both neurotoxicity (Brewer et al., 1994; Schmuck et al., 2002) and resistance (Mbengue et al., 2015; Paloque et al., 2016). This metabolite can be avoided by replacing the C10 oxygen with either a carbon or nitrogen functionality. The group of Jung was the first to synthesize and report the characteristics of such a derivative known as deoxoartemisinin (Figure 1.4) (Jung et al., 1990). It was first synthesized in 1989 and was eight times more active than artemisinin. By removing the unstable acetal functionality, deoxoartemisinin had
6
a half-life of 231.36 hrs in simulated stomach acid compared to artemisinin that only had a half-life of 23.50 hrs (Jung & Lee, 1998). Unfortunately, as with many other derivatives, deoxoartemisinin lacks any toxicity data.
Figure 1.4: Non-acetal derivatives.
Artemisone a C-10 non-acetal alkylamino-artemisinin is a very attractive compound because it avoids the dihydroartemisinin metabolite and lacks neurotoxicity (Figure 1.4) (Haynes et
al., 2006; Schmuck et al., 2003). It enjoys a favourable logP value of 2.49, an extended
half-life compared to the commercially available artemisinins, enhanced anti-plasmodial activity and thermal stability (Haynes et al., 2006; Nagelschmitz et al., 2008). The elimination half-live of artemisone is 5 hours and reaches maximum blood concentrations within 1.5 hours comparable to the clinically used artemisinins (Vivas et al., 2007). Artemisone was 10 times more potent than artesunate (Figure 1.1) against 12 different P. falciparum strains and also 4–10 times more potent than artesunate in rodent models (Vivas et al., 2007). It was found that artemisone was more effective in treating cerebral malaria than artesunate. It was for these reasons that an alkylamino-artemisinin scaffold was used in this study to investigate alkylamino-ferrocene-artemisinin hybrids.
1.2
Aim and objectives
With confirmed resistance towards the artemisinin class there is a need for new artemisinin derivatives to which there is no resistance. To explore new and dual functional hybrid drugs artemisinin-ferrocene hybrids will be investigated. The proposed action of these hybrids would be that after the peroxide functionality artemisinin is destroyed the ferrocene moiety would continue to act as an ROS generator, causing additional damage to the parasite leading to its death. With the ferrocene moiety working independently, it will be a more active drug. Additionally, these derivatives will be coupled together by means of a piperazine linker with the hope that the additional amine functionalities will aid in an improved
7
pharmacokinetic profile for these derivative. Also these non-acetal derivatives will not be metabolised to dihydroartemisinin, eliminating the possible cause of resistance and associated toxicity.
In order to achieve the aim of this study, the following objectives had to be achieved: Synthesis of new amino-artemisinin-ferrocene hybrid derivatives.
The characterisation of these hybrids by means of nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS) and infrared spectroscopy (IR).
Determination of the in vitro antiplasmodial activity of all targeted hybrid derivatives. Determination of the in vitro cytotoxicity of synthesised hybrid derivatives.
8
1.3
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Chapter 2:
Literature review
2.1
Introduction
In 500 B.C., the Romans described a fever called ―bad air‖, better known as malaria today (Hempelmann & Krafts, 2013). They observed that if the water of a swamp or marshland was drained, the number of fever incidences decreased. This created a belief that remained for more than 2000 years that malaria was caused by the vapours and mists from marshes and swamps. Hippocrates, a Greek physician, was able to differentiate between different types of malaria by describing the fevers as quotidian (with a periodicity of 24 hours), tertian (a periodicity of 48 hours) and quartan (a periodicity of 72 hours) (Strong, 1944).
It was not until 1880 that Charles Louise Alphonse Laveran observed parasites in the blood of a patient suffering from malaria (Cox, 2010). Laveran named his severe Summer-Autumn (malignant tertian) malaria Laverania malariae, which would later become known as the malaria caused by the Plasmodium falciparum parasite. The first step towards the differentiation of different types of malaria was made by an Italian neurophysiologist, Camillo Golgi (Golgi, 1886). He postulated that there were at least two types of malaria, namely tertian and quartan. He also observed that the fever of the patient coincided with the release of new parasites into the bloodstream. Four years later, two Italians, Giovanni Batista Grassi and Raimondo Filetti, were the first ones to name the two of the malaria types that affected humans, namely P. vivax and P. malariae (CDC, 2016b). Other species of human malaria are P. knowlesi and P. ovale.
On the 20th of August 1897, the landmark discovery in the field of malaria was made by Ronald Ross (Ross, 1897). The malaria parasite was found in the stomach tissue of an
Anopheline mosquito that previously fed on a malaria patient. Two years later in India, using
malaria in a bird model, he found that the malaria parasites developed inside the mosquitos‘ stomach and then migrated to the salivary glands. This is the pathway that malaria uses to spread: during the blood meals of the mosquito. Thus, the myths surrounding malaria transmission were finally debunked.
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In this chapter, the epidemiology of malaria, the malaria parasite life-cycle and the mechanisms of resistance are briefly discussed. Furthermore, the diagnosis, prevention and control, chemotherapy of malaria, ferrocene and ferrocene-artemisinins are addressed.
2.2
Epidemiology
According to the World Health Organization (WHO), there were approximately 435 000 malaria related deaths worldwide (WHO, 2018). Of these deaths, about 92% occurred in Africa, of which 61% were children under the age of five years old.
There are a number of factors that contribute to the high incidence of the disease especially in Africa. Climate plays an important role. For malaria to be successfully transmitted, the female Anopheles must live long enough to become infected with malaria. Afterwards, the malaria parasite have to undergo the sporogonic cycle, which takes 9-21 days at 25 °C and then cycle within the mosquito to be able to inject the human host with sporozoites species (Wernsdorfer, 2012). It was found that a temperature fluctuation of around < 21 °C speeds up parasite development (Paaijmans et al., 2009). A warmer climate could also increase the human contact with mosquitoes since people may sleep outside or they will be spending more time outside at night. This is why malaria occurs so frequently in sub-Saharan Africa.
The second main reason for the wide occurence of malaria is the type of mosquitoes or vectors. There are mainly two predominant vectors in Africa, namely Anopheles arabiensis and Anopheles coluzzi (Killeen et al., 2017) (Figure 2.1). These Anopheleses are anthropophilic, which means that they prefer to obtain their blood meals from humans. A.
coluzzi are endophagic (preferring to bite indoors) and endophilic (preferring to rest indoors)
while A. arabiensis are mainly exophagic (preferring outdoor biting) and exophilic (preferring to rest outdoors) (Meyers et al., 2016). Although these are the main behaviour patterns for these species, it was found that there was a shift from endophagic to exophagic behaviour when the use of bed nets were introduced (Sougoufara et al., 2014).
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Figure 2.1: Malaria vector species feeding on humans and animals (Killeen et al., 2017).
The last main reason for the large number of fatalities can be gleaned from Figure 2.2 that represents the distribution of P. falciparum, which is prevalent in Africa (WHO, 2010). P.
falciparum malaria is responsible for life-threatening complications (Snow et al., 2005). The
most distinctive complications are cerebral malaria and severe anaemia (Pasvol, 2005). Other manifestations include respiratory distress, renal failure, hypoglycaemia, circulatory collapse, coagulation failure and impaired consciousness.
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2.3
The life-cycle of malaria
The life-cycle (Figure 2.3) of the malaria parasite can be divided into two main parts, namely the human cycle and the mosquito cycle. The human cycle consists of the liver and blood stage . As discussed in § 2.1 with Ross‘ discovery of the malaria sporozoites, these enter the human host through the saliva of the infected female Anopheles when taking a blood meal (Rosenberg et al., 1990). These sporozoites have a limited time (1-3 hrs) to reach the liver before they are no longer motile (Ménard et al., 2008). The sporozoites move through the blood capillaries and interact with the Küpffer cell to enter the liver cell or hepatocyte.
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2.3.1
Human liver stage
The human liver stage is also known as the exo-erythrocytic cycle . In the hepatocytes, the sporozoites matures into forms called schizonts . They then undergo asexual amplification to develop into liver-stage merozoites (Doolan et al., 2009). This process is called exo-erythrotic schizogony and can last for 2-10 days. After 1 to 2 weeks, a schizont can contain up to 30 000 merozoites (Cowman & Crabb, 2002).
When the liver‘s schizont ruptures , the merozoites are released into the bloodstream where they enter red blood cells and begin their erythrocytic stage of their life-cycle (Doolan
et al., 2009). However, not all of the Plasmodia follow the same route. P. vivax and P. ovale
do not all form mezorites, but they form some hypnozoites. These are dormant and can remain that way for months, or even years. They can then generate merozoites, which cause a relapse of malaria (Cogswell, 1992).
2.3.2
Human blood stage
The erythrocytic stage begins when the merozoites infect red blood cells (RBCs) . The merozoite gains entry by attaching itself to RBC . Reorientation follows so that the apical end can form a tight junction with the RBC. From here, it moves into the RBC and finally reseal the RBC membrane (Farrow et al., 2011). Here, the merozoite flattens out into the immature trophozoite/ring stage. Its diet mainly consists of the cytosol, by endocytosis, as a source of essential amino acids and haemoglobin (Bannister et al., 2000).
The ingested haemoglobin is broken down to ferriprotoporphyrin IX and is toxic due to its ability to induce redox cycling and to generate a reactive oxygen species (ROS) (Dassonville-Klimpt et al., 2011; Kumar & Bandyopadhyay, 2005). The ferriprotoporphyrin IX mainly becomes detoxified inside the parasite‘s food vacuole where the acidic conditions, namely a pH of 5.2, promote the formation of hemozoin (malaria pigment) (Dassonville-Klimpt et al., 2011; Kumar & Bandyopadhyay, 2005). The trophozoites undergo nuclear division forming schizonts and producing new merozoites in the RBC. Finally, the RBC ruptures and the merozoites are released into the bloodstream, ready to infect new RBCs (Bannister et al., 2000). After a few of these cycles, some of these merozoites develop into male and female gametocytes . The gametocytes circulate in the peripheral circulation and are ingested by the Anopheline mosquito when it takes a blood meal (Kuehn et al., 2010).
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2.3.3
Sporogonic cycle
This cycle begins when the Anopheline ingests blood infected with gametocytes (Kuehn et
al., 2010). After ingestion by the mosquito, the male and female gametocytes are released
from their red blood cells in response to environmental changes, including temperature and pH changes (Sinden et al., 1996). In the midgut of the mosquito, the gametocytes mature into gametes. The male gametocytes undergo division and develop flagella which turn them into motile micro gametes. These fertilise the female macro gametes to form zygotes , then ookinetes , and then they mature into oocysts . Within a few days, the oocysts rupture and release thousands of sporozoites which collect within the salivary glands of the mosquito – ready to infect the next human host (Touray et al., 1992).
2.4
Pathology
After a person has been infected with malaria, symptoms occur within 10–21 days. These symptoms are the result of the rupturing of the erythrocytes (Malaguarnera & Musumeci, 2002). Initially, the symptoms manifest themselves as headaches, slight fever, muscle pain and nausea – much like flu symptoms. This phase is followed by febrile attacks, also known as paroxysms (James et al., 1936). The paroxysms appear in three different stages. During the cold stage, the person experiences intense feelings of cold and shivering that last between 15–50 minutes. The heat stage is characterised by feelings of intense heat, dry burning skin and a throbbing headache which lasts between 2–6 hours. Lastly, this is followed by the sweating stage where the person experiences profuse sweating, a decline in body temperature and exhaustion, leading to sleep – this stage lasts between 2–4 hours (Alvarez et al., 2005). The time between these paroxysms are also indicative of the type of malaria: 24 hours for P. knowlesi, 48 hours for P. falciparum, P. vivax and P. ovale and 72 hours for P. malariae. The most dangerous of the Plasmodium species is P. falciparum. The main reason for this is due to the high levels of parasitaemia which lead to a higher level of destruction of the erythrocytes (Jakeman et al., 1999). These levels give rise to severe malaria in 90% of cases. Complications experienced are renal failure, respiratory distress, hypoglycaemia, circulatory collapse, coagulation failure and impaired consciousness. The most dangerous complications that can occur is cerebral malaria and severe anaemia (Goldsmith, 1997).
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2.5
Diagnosis
Malaria is diagnosed mainly by microscopy or by a rapid diagnostic test (RDT). Microscopy is the most widely used diagnostic method and has a detection limit of 250 parasites/µL for thin smears (Fançony et al., 2013; Harchut et al., 2013). The main limitation of this method is that it requires a highly trained technician which may become fatigued due to the frequent high workload associated with this profession (Ansah et al., 2010; Reyburn et al., 2007). This can be overcome by the use RDTs – a method that requires minimal training. RDTs work by detecting parasite specific antigens, but these may be affected by residual parasite antigens. Also, the consistency of RDTs varies between brands and batches (Alonso & Tanner, 2013; Mouatcho & Goldring, 2013).
More recently, an analytical tool called the Sight Diagnostic Parasight platform is under development and might be the future of malaria diagnosis. The detection limit for this device is currently as low as 20 parasites/µL with future updates being as low as 5 parasites/µL (Eshel et al., 2017). Unfortunately, the device is currently unable to distinguish between P.
vivax and P. ovale – but fortunately, the treatments for these parasites are the same. P. falciparum identification is, however, highly specific.
2.6
Control and prevention
The two most commonly used methods for the prevention of malaria are insecticide-treated nets (ITN) and indoor residual spraying (IRS). It is estimated that these two methods have helped to prevent 663 million cases of malaria in Africa alone (Cibulskis et al., 2016). Although mosquitoes have developed some resistance to the insecticides used on ITNs, these still prevent biting during night-time use.
Currently, there are numerous new strategies formulated to combat the mosquito vector. Attractive toxic sugar baits (ATSBs) are part of the lure- and kill-strategy being evaluated. A 10% sucrose solution combined with boric acid or ivermectin is used in bait stations or is sprayed on vegetation (Barreaux et al., 2017; Tenywa et al., 2017). In field trials it was found that these ATSBs killed up to 90% of the mosquito population (Qualls et al., 2015). Eave tubes are another part of the lure- and kill-stratagem. These tubes are simply PVC tubes covered with an insecticide-treated mesh net. As the mosquitoes try to enter the house through the tubes, they come in contact with the electrostatic insecticide on the mesh. The
22
insecticide is then transferred to the mosquito, which leads to its death (Andriessen et al., 2015).
The behaviour of mosquitoes while mating could also be exploited. They form swarms, not higher than 3m above the ground, which could easily be sprayed with an insecticide (Diabate & Tripet, 2015). Endectocides that target mosquitoes while taking a blood meal are also being field tested, but these have short half-life times and there is a lack of understanding of the mechanism of action. Furthermore, spatial repellents ensure an environment that is free of mosquitoes – thus helping to decrease malaria transmission. One such compound is dichlorodiphenyltrichloroethane (DDT). The success of DDT is primarily attributed to it being a spatial repellent rather than being a toxic substance. This kind of substance modifies the behaviour of the mosquitoes, and currently the debate is on whether or not these should be a toxic or an irritant substance. This issue requires further investigation.
Lastly, vaccination may be an option. Over the last 10 years, there were at least 40 different vaccines that reached clinical trials. The RTS,S/AS01 vaccine, targeting the pre-erythrocytic stage, is the only vaccine that shows promise and is recommended for pilot implementation studies in Africa. During the phase 3 trials, it showed a 45.7% protection among infants over an 18 month period after 3 vaccination doses.
2.7
Chemotherapy
This chapter also introduces the main antimalarial drugs with a focus on the use of these as partner drugs in antimalarial combinational therapy (ACT). The currently used antimalarials are divided into five main pharmacological classes, namely quinoline and quinoline-based antimalarials, antifolates, antibiotics, hydroxynapthoquinones and, lastly, the artemisinin class.
23
2.7.1
Quinoline and quinoline based antimalarials
2.7.1.1 Aryl-amino alcohols
QuinineFigure 2.4: Quinine.
The bark of the fever tree (cinchora tree) has been used to treat fever since the Inca civilisation. One of the active compounds found in this bark was quinine. The bark of the cinchora tree, which was later exported to treat malaria in Europe (Bruce-Chwatt, 1988). Although the isolation of this active alkaloid is controversial it was either in Germany in 1819 or 1820 by French chemists (Meshnick & Dobson, 2001). The first successful synthesis was reported in 1944 by American chemists. This late finding is ascribed to the fact that the supply of cinchona bark to America was cut off because of the Japanese presence in the south Pacific during World War II (Schlitzer, 2007; Wacks, 2013; Woodward & Doering, 1945). Quinine is an active blood schizonticide against P. falciparum, P. malaria, P. ovale and P. vivax and it is also somewhat active gametocytocidal against P. malaria and P. vivax (Murambiwa et al., 2011).
Resistance to quinine probably emerged due to the short half-life of only 8–10 hours. It is generally accepted that quinine accumulates in the parasites‘ acidic digestive food vacuole and inhibits haemozoin biomineralisation (Fitch, 2004). A decrease in sensitivity towards quinine was reported in Brazil in 1910 (Björkman & Phillips-Howard, 1990; Meshnick, 1997), and in some parts of Asia its efficacy has fallen below 50% (Giboda & Denis, 1988). It was found that the main 3 genes responsible for quinine resistance are Pf chloroquine resistance transporter (PfCRT), Plasmodium falciparum multidrug-resistance gene 1 (PfMDR1) and Plasmodium falciparum Na+/H+ Exchanger Gene (PfNHE1) (Cooper et al., 2002; Cooper et
al., 2007; Nkrumah et al., 2009; Sidhu et al., 2002).
Quinine is still widely used as a monotherapeutic drug in Africa due to its affordability (Watsierah & Ouma, 2014). The quinoline-based antimalarials are mefloquine, amodiaquine, primaquine, halofentrine and lumefantrine.
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Mefloquine
Figure 2.5: Mefloquine.
Mefloquine was discovered between 1963–1976 when the USA army launched a drug discovery programme during the Vietnam War (White, 1992). Mefloquine is a 4-methanolquinoline, with blood schizonticidal activity against the asexual stages of P.
falciparum and P. vivax. Mefloquine reaches peak concentrations within 24 hours. and has
an elimination half-life of 2–3 weeks (Stepniewska & White, 2008). It can be used as a prophylactic drug, but it has neuropsychiatric side effects such as psychosis, seizures, hallucinations and vertigo (Weinke et al., 1991).
Resistance to mefloquine was first noted in Thailand in 1982 (Boudreau et al., 1982). Resistance have also been reported in Africa, and this might be due to quinine resistance (Oduola et al., 1988; White, 1994). The P. falciparum develops resistance to mefloquine by the amplification of the PfMDR1 gene and the over-expression of its protein product Pgh-1 (Cowman & Crabb, 2002; Peel et al., 1993; Wilson et al., 1993).
Halofantrine
Figure 2.6: Halofantrine.
As is the case with mefloquine, halofantrine was discovered in the 1960‘s during the Vietnam War by the Walter Reed Army Institute of Research (Ugochukwu et al., 2008). It reaches peak plasma concentrations within 4–8 hrs, and has an elimination half-life of 3–7 days for the active metabolite (de Villiers et al., 2008). The use of halofantrine has been withdrawn due to the significant risk of death resulting from ventricular tachyarrhythmia. As with the previous drugs, mutations of the PfMDR1 gene are responsible for causing resistance. It has
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been reported that the mutations of this gene modified the transport of halofantrine, and this might be indicative of being a mechanism of resistance to this drug (Sanchez et al., 2008).
Lumefantrine
Figure 2.7: Lumefantrine.
Lumefantrine is structurally related to quinine and mefloquine, and therefore it is believed that the mechanism of action should be similar to these (Alin et al., 1999). Although lumefantrine induces fewer cardio-cytotoxic side-effects, it is not without its problems (van Agtmael et al., 1999). Slow absorption, low bioavailability, and having weaker antimalarial activity than halofantrine indicate that lumefantrine cannot be used as monotherapy (White
et al., 1999). However, the bioavailability can be increased by as much as a factor of 16
when taken with a fatty meal. The terminal elimination half-life varies between 30–107 hours. Mutations in the PfMDR1 gene are associated with lumefantrine resistance (Nzila et al., 2012).
2.7.1.2 4-Aminoquinolines
ChloroquineFigure 2.8: Chloroquine (CQ).
Chloroquine is a derivative of quinine and is the most widely used antimalarial drug. It was first synthesised in 1934 by Hans Andersag and his co-workers at Bayer laboratories under the trade name Resochin (Savarino et al., 2003). Chloroquine is a schizonticide against chloroquine-sensitive P. falciparum and is used in areas with predominant P. vivax
